Antibody molecules are formed by the association of two heavy (H) chains and two light (L) chains by means of disulphide bridges. The two heavy chains are mutually associated in accordance with a Y-shaped structure and the two light chains respectively associate with the two branches of this structure in such a way that the variable domains of the light chains (VL) and heavy chains (VH) are-situated close to each other. The binding to the antigen results from the properties of the variable parts of the light and heavy chains. A complex system of rearrangement and selection then enables a substantial quantity of antibody which is specific to an antigen to be induced rapidly.
The standard hybridoma technique makes it possible to select clones of hybrid cells which express genes encoding the light and heavy chains of an antibody molecule. This technique requires the fusion of cells of lymphocytic origin, which contain genes directing the formation of antibodies, and of cells which are capable of giving rise to hybridomas which form immortalized cell lines. The cells which carry the genes in question are generally obtained by randomly creating libraries of circulating cells, with or without prior immunization with a specific antigen, and the hybridomas are screened by means of an antigen-antibody reaction after multiplying and culturing the hybridoma clones. This technique is cumbersome, with a limited yield, and the screening is not simple.
Another method, which uses recombinant bacteriophages, has recently been employed. The principles and the various ways of carrying out this method are described, for example, by D. R. Burton, Tiptechxe2x80x94May 1991, vol. 9, 169-175; D. J. Chiswell et al., Tiptechxe2x80x94March 1992, vol. 10, 80-84; H. R. Hoogenboom et al., Rev. Fr. Transfus. Hxc3xa9mobiol., 1993, 36, 19-47 (see, also, Patent Applications PCT WO 92/01047 and 92/20791).
This technique consists in inserting, into a vector, a repertoire of genes for variable regions of antibodies in association with a bacteriophage gene under conditions which enable the gene to be expressed in the form of a fusion protein which is presented at the surface of the phage, thereby exposing the variable regions of the light and heavy variable chains, which are linked by their disulphide bridges in the manner of an antibody Fab fragment, and in selecting the phages directly by means of a rapid separation method employing the immobilized specific antigen, for example by means of immunoaffinity chromatography. The selected phages can, following elution, infect a bacterium and be used directly for production or for repeating selection cycles. This method is particularly powerful since, in theory, it enables very substantial libraries to be created and a library to be screened in an extremely discriminating, efficient and rapid manner. A phage which lends itself particularly well to this method is the filamentous phage fd, in which it is possible to fuse the fragment encoding one of the heavy or light chains of the antibody with the gene for the minor surface protein and to insert the fragment encoding the other chain in such a way that, after the phage has infected bacteria, a population of phages is obtained which carry, at their surface, a fusion protein which presents the heavy and light chains in a configuration which is able to recognize the antigen and which can, therefore, be screened.
In addition to its simplicity, this technique offers great advantages. In association with the preliminary amplification of the library of antibody genes, it is possible to select one phage presenting a specific antibody fragment in a very large population of phages, of the order of 107, which can make it possible to search for human antibody genes without having necessarily to carry out a prior immunization of the donor.
By means of cleavage followed by religation, or by using two separate libraries of genes for light chains and for heavy chains, it is possible to obtain phages linking one light chain and one heavy chain in a random manner.
However, the number of different clones which it is possible to obtain is limited by the yield of the selection and by the degree of efficiency of the bacterial transformation.
A means of increasing the number of successful combinations linking the light chains of a first library to the heavy chains of a second library was described by P. WATERHOUSE et al., Nucleic acids Research, 1993, Vol. 21, No. 9, 2265-2266, enabling up to 1012 clones to be obtained using a loxP site-specific recombination system which is sensitive to the action of a Cre recombinase. However, the linkage. remains reversible. Furthermore, there is no control over the action of the recombinase and the recombined vectors do not have any selective advantage over the other vectors.
Now, taking into account the yield of the step for selecting recombinant phages which, in reality, only retains a fraction of the phages of interest, it is desirable to obtain the highest possible yields of recombinant vectors with the least possible number of non-recombinant vectors.
An object of the present invention is, therefore, to provide a process for producing multicombinatorial libraries, in particular in the form of phages or phage-mids, from two repertoires of genes, one for light chains and the other for heavy chains, enabling a very high number of clones to be obtained.
Another object of the invention is to provide such a process in which the number of non-recombinant vectors which are present at the end of the process is reduced.
Another object of the invention is to produce recombinants of this nature which have an enhanced stability.
The invention relates to a process for producing multicombinatorial libraries in which, starting from a first repertoire of genes encoding a population of one of two types of polypeptides which are capable of combining with each other, covalently or non-covalently, in particular variable regions of one of the light chain and heavy chain antibody types, and from at least one gene encoding the other type of polypeptide, in particular a variable region of the other type of antibody chain, or preferably from a second repertoire of genes encoding a population of the said other type, the genes of the said first repertoire are introduced into a first vector in order to form a population of vectors carrying the different genes of the said first repertoire, and the said gene of the said other type or the genes of the said second repertoire are introduced into a second vector, at least one of the said vectors, termed recipient, being arranged to receive, by recombination with the other vector, all or part of the said other vector, together with its gene, under conditions enabling the said recipient vector to contain, after recombination, a gene of one of the two types and a gene of the other type and to express the two genes in the form of linked polypeptides which are able to appear on the external surface of the product of the said vector, being maintained there and being linked together in a multimeric manner, or simulating a multimer, characterized in that the said vectors exhibit means enabling the recombination of the two types of chains to be effected in an irreversible manner.
In that which follows, the invention will be described in the application in which the two types of polypeptides are regions, which are at least in part variable, of light and heavy chains of antibodies. However, it will be understood that the invention also applies to other types of polypeptides which are capable of combining with each other, in particular the chains of heterodimeric receptors such as the xcex1 and xcex2 chains or xcex3 and xcex4 chains of the T-cell receptors.
Particularly advantageously, the vectors, which are preferably circularized, respectively contain the E. coli attB and the xcex phage attP specific recombination sites which are arranged in such a manner as to permit recombination under the influence of the associated recombinase or integrase thereby forming, in a single vector resulting from the recombination, stable junction sequences such as attL and attR.
Naturally, it would also be possible to replace these sites by the specific recombination sites of other bacterium/phage, or alternative, recombination systems.
In order to enable vectors which only contain a single chain to be eliminated and an additional increase in the relative content of recombinant vectors to be obtained as a consequence, the said vector can be arranged to have a selection marker which is initially non-functional and which is rendered functional by the recombination.
Preferably, the selection marker comprises a gene which permits selection, when it is expressed, and a promoter which is specific for the gene, the promoter being inserted into one of the vectors and the marker gene being inserted into the other, in such a way that they become linked in the recombinant vector which is obtained. The preferred markers include the genes, together with their promoters, for resistance to chloramphenicol, to tetracyclines and to gentanycin.
Naturally, the two vectors can also contain specific markers which can be used in the specific construction of each of the vectors, so as to permit or improve selection of each of the vectors during the process of its construction. As an example, these markers can be the genes for resistance to antibiotics such as kanamycin and ampicillin.
Preferably, a thermo-inducible recombinase is used to control the recombination step between the first and the second vector, for example by means of choosing a suitable host such as the E. coli strain D1210 HP, which is to be found in the catalogue of the American company Stratagene.
The invention also relates to the vectors which are obtained by the process according to the invention, in particular the vectors of the plasmid or phagemid type, which are characterized by the presence of a sequence encoding a variable part of an antibody light chain, and a sequence encoding a variable part of an antibody heavy chain, the said sequences. being accompanied, in suitable environments, by elements which enable them to be expressed in a host, the said light-chain and heavy-chain sequences, together with the said means which are linked to them, being respectively separated by unique irreversible integration sites such as, in particular, attP or attB.
Finally, the invention relates to multicombinatorial libraries which are formed by the vectors according to the invention and which combine, in a random manner, a sequence encoding one of the polypeptide types, such as a variable part of a light chain, and a sequence encoding the other polypeptide type, such as a variable part of a heavy chain.
An example will now be described of the construction of a recombinant vector according to the invention which combines the variable parts of the light chain and the heavy chain of one and the same anti-HIV gp160 clone, a vector which is found to be able to express the genes of the said light and heavy variable parts and to present their expression products at its surface or, as a variant, to release them in the form of a heavy chain-light chain combination which recognizes the gp160 antigen.